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. 2002 Aug;106(4):526–536. doi: 10.1046/j.1365-2567.2002.01442.x

Limitations of HLA-transgenic mice in presentation of HLA-restricted cytotoxic T-cell epitopes from endogenously processed human papillomavirus type 16 E7 protein

Michael D Street 1,*, Tracy Doan 1,*, Karen A Herd 1, Robert W Tindle 1
PMCID: PMC1782761  PMID: 12153516

Abstract

We investigated the use of mice transgenic for human leucocyte antigen (HLA) A*0201 antigen-binding domains to test vaccines composed of defined HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitopes of human papillomavirus (HPV) type 16 E7 oncoprotein. HPV is detected in >90% of cervical carcinomas. HPV16 E7 oncoprotein transforms cells of the uterine cervix and functions as a tumour-associated antigen to which immunotherapeutic strategies may be directed. We report that although the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7-directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization-introduced E7, by the mouse antigen-processing and presentation machinery. Thus (1) CTL induced by HLA A*0201 peptide immunization killed E7 peptide-pulsed target cells, but did not kill target cells expressing whole E7; (2) immunization with whole E7 protein did not elicit CTL directed to HLA A*0201-restricted E7 CTL epitopes; (3) HLA A*0201-restricted CTL epitopes expressed in the context of a DNA polytope vaccine did not activate E7-specific T cells either in ‘conventional’ HLA A*0201 transgenic (A2.1Kb) mice, or in HHD transgenic mice in which expression of endogenous H-2 class 1 is precluded; and (4) HLA A*0201 E7 peptide epitope immunization was incapable of preventing the growth of an HLA A*0201- and E7-expressing tumour. There are generic implications for the universal applicability of HLA-class 1 transgenic mice for studies of human CTL epitope presentation in murine models of human infectious disease where recognition of endogenously processed antigen is necessary. There are also specific implications for the use of HLA A2 transgenic mice for the development of E7-based therapeutic vaccines for cervical cancer.

Introduction

Mice in which human leucocyte antigen (HLA)-class 1 transgene products function as restriction elements for cytotoxic T-lymphocyte (CTL) responses for ‘human’ CTL epitopes in viral proteins are frequently used as immunological models of human viral disease and vaccine development.1–4 The HLA transgenic mouse models generally assume conserved antigen processing and presentation of antigen to generate the appropriate ‘human’ 8–10 mer CTL epitopes, physiological peptide selection by human class 1 molecules in murine antigen presentation cells, and an appropriate CD8+ T-cell repertoire containing T-cell receptors (TCRs) capable of positive selection on the ‘human’ epitope-HLA class 1 complex. While recent reports5 suggest that all components of the antigen presentation machinery may not function normally across species barriers (or even within class 1 polymorphisms within species), nonetheless HLA class 1 (particularly HLA A*0201) transgenic mice have been used to identify CTL epitopes subsequently shown to be functional in human TCR- and antigen-processing systems.1,6–9

We have studied the potential use of mice transgenic for HLA A*0201 antigenic binding domains as a putative model in which to test vaccines designed to induce HLA A*0201-restricted CTL responses to the E7 oncoprotein of human papillomavirus type 16 (HPV16). HPV DNA (particularly that of HPV16) is detected in >90% of cervical carcinomas. The E7 oncoprotein persists in transformed cervical epithelial cells10 and thus E7 functions as a tumour-associated antigen to which immunotherapeutic strategies designed to elicit tumour-controlling cytotoxic T-lymphocyte responses may be directed.3,11–16 CTL have been shown to exert anti-tumour activity in humans.17,18

Several HLA A*0201-restricted E7 CTL epitopes have been identified by peptide–major histocompatibility complex (MHC) binding algorithms.7,19 These epitopes are processed and presented from whole E7 for both induction of primary CTL responses and for susceptibility to lysis by appropriate CTLs in A*0201+ humans.19,20 Furthermore, these epitopes have been shown to elicit CTL responses when used as peptide vaccines in HLA A*0201 transgenic mice. In the present study we report the following.

While immunization of HLA A*0201 transgenic mice with these peptide epitopes will elicit CTL, immunization with whole E7 protein will not elicit CTL capable of killing peptide-pulsed HLA A*0201 target cells.

While CTL induced by HLA A*0201 peptide epitope immunization will kill peptide-pulsed target cells, they will not kill murine target cells expressing whole E7.

HLA A*0201-restricted CTL epitope expressed in the context of a DNA polytope vaccine will not activate E7-specific T cells either in ‘conventional’ HLA A*0201 transgenic (A2.1Kb) mice, or in HHD A*0201 transgenic mice in which expression of endogenous H-2 class 1 is precluded.

HLA A*0201 peptide epitope immunization is incapable of preventing the growth of an HLA A*0201- and E7-expressing tumour.

We conclude that while the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7-directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization-introduced E7, by the mouse antigen-processing and presentation machinery. A corollary is that the concept of the universal applicability of HLA transgenic mice as ‘human’ models of specific immunity may be flawed if cross-species incompatibility in antigen-processing and presentation machinery precludes or skews the presentation of some HLA-restricted epitopes.

Materials and methods

Mice

A2.1Kb mice express a chimeric HLA class 1 molecule, A2.1Kb, composed of the α-1 and α-2 domains of HLA A*0201, and the α-3 transmembrane and cytoplasmic domains of H-2Kb,4 on a predominantly C57BL (H-2b) background. A2.1Kb mice are capable of making CTL responses restricted through both HLA A*0201 and H-2b class 1 molecules. HHD mice express a transgenic monochain class 1 molecule in which the C-terminus of human β2-microglobulin is covalently linked to the N-terminus of a chimeric heavy chain (α-1 and α-2 of HLA A*0201, and α-3 of H-2Db).21 The H-2Db and mouse β2-microglobulin genes have been disrupted. Thus, HHD mice are capable of HLA A*0201-restricted CTL responses, but not H-2-restricted CTL responses. Founder mice from both lines were derived by Caesarean section, and all mice were housed under specific pathogen-free conditions. Genetic authenticity was confirmed at intervals by polymerase chain reaction (PCR) for the transgene. Mice were used at 7–15 weeks of age, but within a given experiment were littermates or closely age- and sex-matched.

Peptides, proteins

Peptides representing defined HPV16 E7- and influenza virus matrix protein-CTL epitopes (Table 1) were synthesized with free ends using 9-fluorenylmethoxycarbonyl (F-moc) chemistry and analysed by high-performance liquid-chromatography (HPLC) by Chiron Corporation (Melbourne, Australia). Peptide stocks were made at 10 mg/ml in dimethyl sulphoxide and diluted into tissue culture medium for assays. HPV16 E7 was expressed in Escherichia coli as a glutathione-S-transferase fusion protein. E7-enriched inclusion bodies were solubilized in 10 m urea, and the protein was purified using anion exchange chromatography, as described previously.22

Table 1. Peptide epitopes.
Peptide Sequence* Origin HLA restriction Reference
E7/A2/1 86TLGIVCPI93 HPV16 E7 HLA A*0201 19
E7/A2/2 82LLMGTLGIV90 HPV16 E7 HLA A*0201 19
E7/Db 44QAEPDRAHYNIVTFCCKCD62 HPV16 E7 H-2Db 42
Influenza/A2 58GILGFVFTL66 Influenza virus matrix protein HLA A*0201 43
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*

Minimal epitope is underlined.

Cells

EL4.E7 cells were derived by transfection of EL4 cells with a plasmid encoding a full-length HPV16 E7 gene as described23. Expression of E7 was confirmed by dot blot hybridization of a 32P end-labelled E7 oligonucleotide probe and by specific lysis by a T-cell line specific for the immunodominant H-2Db-restricted E7 CTL epitope 49RAHYNIVTF57.23 EL4.A2 and EL4.E7.A2 cells were prepared by transfection of EL4 and EL4.E7 cells, respectively, with plasmid A2.1Kb encoding the chimeric MHC class 1 heavy chain (above)4, as described previously.16 HLA A*0201 cell surface expression was measured in EL4.A2 and EL4.A2.E7 cells using the monoclonal antibody (mAb) MA2.1 and H-2Db expression was measured using mAb 28-8-6, in indirect immunofluorescence with F(ab′)2-anti-mouse immunoglobulin G (IgG)/fluoroscein isothiocyanate (FITC; Caltag, Burlington, CA) secondary reagent and flow cytometry (FACS IV, Becton Dickinson & Co, Sydney, Australia). EL4.A2 and EL4.A2.E7 cells are susceptible to specific CTL lysis through both the H-2b and the HLA A*0201 restriction pathways. EL4S3– RobHHD cells24, negative for endogenous MHC and transfected with the HHD construct, were maintained under G418 selection. TC-1.A2 cell line was derived from the E7-expressing epithelial tumour cell line TC-1,25 by transfection with plasmid A2.1Kb, and expression of HLA A*0201 α-chains was confirmed as above. Cells were maintained in RPMI medium (Gibco Melbourne, Australia) supplemented with 2 mm glutamine, 1 mm sodium pyruvate, 20 mm HEPES, 5×10−5mβ-mercaptoethanol, 100 IU/ml penicillin, 100 µg/ml. streptomycin and 10% fetal bovine serum.

Immunization of mice and restimulation of splenocytes for CTL

In standard CTL induction experiments, mice were immunized subcutaneously (s.c.) at the tail base with 50 µg peptide + 0·25 µg tetanus toxoid (TT) as a source of T-helper epitopes + 10 µg Quil A adjuvant,26 or with 50 µg E7.GST fusion protein+Quil A. Ten days later spleens were removed and splenocytes were restimulated in vitro for 6 days in 24-well tissue culture plates (5×106 cells per well) in the presence of 1 µg/ml cognate peptide. (Note that immunization alone, or in vitro stimulation alone does not generate measurable CTL in these experiments). In some experiments splenocytes were restimulated with irradiated (20 000 rads) EL4.A2.E7 tumour cells at a splenocyte to tumour cell ratio of 10 : 1. For DNA immunizations, anaesthetized mice were injected twice at 2-weekly intervals intradermally (i.d.) with 10 µg, or intramuscularly (i.m.) in each quadriceps with 50 µg of purified plasmid DNA. Three weeks later, splenocytes were harvested for peptide restimulation. In the case of HHD mice, restimulations were performed by co-culturing splenocytes with lipopolysaccharide-induced syngeneic lymphoblasts loaded with peptide at 10 µg/ml.21

CTL assay

CTL assays were conducted as previously described.27 In summary, target cells (104 per well) sensitized at 37° for 1 hr with 1 µg/ml cognate or irrelevant peptide, or medium alone, or expressing the E7 gene, and labelled with 100 µCi 51Cr, were incubated with effector cells at various effector to target cell ratios in triplicate in 96-well microtitre plates. Negative controls included wells containing target cells but no effector cells (i.e. background). Supernatants were harvested from CTL assays at 4 hr, and 51Cr release was quantified by gamma-counting. Results are expressed as per cent cytotoxicity ±SD [calculated as (51Cr release in experimental wells – background)/(detergent-mediated total release – background)×100%].

MHC class 1 tetramers and FACS staining

Phycoerythrin (PE) -conjugated HLA A*0201 tetramers of epitopes E7/A2/2 and influenza/A2 were constructed by the NIH Tetramer Facility (Atlanta, GA). For staining, splenocytes or post in vitro restimulation cells were separated over Ficoll-Paque (Pharmacia, Uppsala, Sweden), and 106 leucocytes were co-stained with tetramer and FITC-anti-mouse CD8a (Sigma Chemicals, Castle Hill, Australia) at 4° for 60 min, prior to washing, fixation in 1% paraformaldehyde and FACS analysis. Tetramer dilutions and staining conditions were optimized in preliminary experiments. Data sets included staining with ‘irrelevant’ MHC-matched tetramer, and staining of cognate tetramer on ‘irrelevant’ CD8+ T cells as negative controls. FACS plots record 60 000 events. Cells positive for both tetramer and CD8 staining are expressed as a percentage of total CD8+ cells.

Tumour protection

Tumorigenicity of TC-1.A2 was assessed by s.c. injection of graded doses of cells in the flank of A2.1Kb mice. Tumour challenge doses in subsequent protection studies were selected to be minimal for tumour induction. Persistence of the A2.1Kb and E7 genes in TC-1.A2 cells in vivo was confirmed by PCR using A2.1-specific primers and E7-specific primers, respectively, of DNA extracted from tumour biopsies 12 days after tumour cell injection. Groups of five mice were immunized twice s.c. with 50 µg peptide + TT + Quil A, or phosphate buffered saline (PBS) and challenged with TC-1.A2 cells s.c. in the flank 2 weeks after the last immunization. Tumour growth was quantified as per cent mice free of palpable tumour at intervals to 60 days after tumour injection.

DNA polytope

The pCCV2000 was constructed essentially as described28 by inserting into the plasmid pDNAVacc, DNA encoding an artificial polytope protein containing an endoplasmic reticulum signal sequence, the PADRE T-helper epitope29 and a number of CTL epitopes including the HLA A*0201-restricted epitopes E7/A2/1 and E7/A2/2, the H-2Db-restricted epitope E7/Db of HPV16E7 and the HLA A*0201-restricted epitope influenza/A2. Authenticity of pCCV2000 was confirmed by DNA sequencing. Plasmid DNA for DNA vaccination was prepared using a Qiagen EndoFree plasmid kit (Qiagen, Clifton Hill, Australia).

Results

EL4-derived A2.1-transfected target cells express HLA A*0201 peptide binding domains

To produce murine target cells for evaluating HLA A*0201-restricted CTL responses, we transfected EL4 and EL4.E723 cells with A2.1Kb plasmid,4 to derive EL4.A2 and EL4.E7.A2 cells, respectively. We showed that EL4.A2 and EL4.E7.A2 expressed high levels of A2.1 at the cell surface and that all the EL4-based lines expressed H-2Db at the cell surface, by indirect immunofluorescence (Fig. 1).

Figure 1.

Figure 1

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MHC class 1 expression in EL4-derived cell lines A2.1-and H-2Db expression were measured at the cell surface of cells by staining with HLA A*0201- and H-2Db-specific antibodies and flow cytometry. Negative controls included staining with irrelevant antibody (shown only for H-2b staining).*E7 expression was confirmed by susceptibility to specific lysis by a CTL line specific for the HPV16 E7 H-2Db-restricted epitope.23

Murine CTL recognizing HLA A*0201-restricted E7 epitopes kill A2.1-target cells pulsed with E7 peptide

In a preliminary experiment, we first confirmed that the line of A2.1Kb mice used in our experiments was effective in mounting HLA A*0201-restricted CTL responses. Restimulated splenocytes from A2.1Kb mice immunized with a model HLA A*0201-restricted CTL epitope peptide (influenza/A2, Table 1) showed a high level of killing of EL4.A2 cells pulsed with influenza/A2 peptide, but not of unpulsed EL4.A2 cells (Fig. 2a).

Figure 2.

Figure 2

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Splenocytes from A2.1Kb mice immunized with influenza/A2 (a), E7/A2/1(b), E7/A2/2(c), or E7/Db(d) peptides were restimulated with cognate peptide and reacted with EL4.A2 or EL4 target cells pulsed with cognate peptide as indicated, in a 4-hr 51Cr-release assay. Negative controls were lysis of EL4.A2 without peptide (as shown) or pulsed with irrelevant peptide (<15% at an effector to target ratio of 50 : 1).

We then wished to confirm the ability of E7/A2/1 and E7/A2/2 HLA*0201-restricted epitopes of HPV16 E7 (Table 1) to elicit CTL responses in A2.1Kb mice. Splenocytes from A2.1Kb mice immunized and restimulated with E7/A2/1 or E7/A2/2 peptides specifically killed EL4.A2 target cells pulsed with E7/A2/1 or E7/A2/2 peptides, respectively (Fig. 2b, c). EL4 cells (which do not express A2.1) pulsed with these peptides were not killed at a level above background (not shown), indicating that specific cytotoxicity was restricted through A2.1. The percentage target cell kill of E7/A2/2 peptide-pulsed EL4.A2 cells was similar to that of E7/Db peptide-pulsed EL4.A2 cell by splenocytes from mice immunized and restimulated with E7/Db peptide containing the murine H-2Db-restricted E7 epitope (Fig. 2d). In addition, titration showed that the amounts of peptide required to sensitize target cells for killing through the HLA pathway were comparable to those required for killing through the H-2 pathway (Fig. 2c, d). These data suggest that killing through the HLA A*0201 pathway was not limited by the level of expression of A2.1 in EL4.A2 cells.

We also demonstrated comparable levels of killing of EL4 cells and EL4.A2 cells by E7/Db-directed CTL (Fig. 2e), indicating that incorporation of the A2.1Kb gene had not altered the susceptibility of EL4.A2 cells to CTL-mediated lysis.

Overall, these data confirm the efficacy of E7/A2/1 and E7/A2/2 peptides as HLA A*0201-restricted immunogenic CTL epitopes in A2.1Kb mice.

Murine CTL recognizing HLA A*0201-restricted E7 epitopes fail to kill endogenously E7-expressing A2.1 murine target cells

CTLs which effectively killed E7-peptide-pulsed EL4.A2 cells in the above experiment were concomitantly evaluated for their ability to kill cells endogenously expressing E7. CTLs directed to E7/A2/1 and E7/A2/2 epitopes failed to kill EL4.A2.E7 target cells which constitutively express E7 and A2.1 (Fig. 3a, b). These data suggest that EL4.A2.E7 cells are incapable of processing E7 for presentation on the cell surface of E7/A2/1 and E7/A2/2 epitopes in the context of A2.1. By contrast, CTLs directed to E7/Db showed killing of EL4.A2.E7, comparable to Db-restricted killing of EL4.E7 (Fig. 3c). These latter data indicate that the apparent inability of EL4.A2.E7 cells to present HLA A*0201-restricted epitopes was not due to a constitutive inability of these cells to process E7. Furthermore, we eliminated the possibility that EL4.A2.E7 cells were not susceptible to lysis through the HLA A*0201 pathway by showing that EL4.A2 E7 cells pulsed with a 0·01–1 µg/ml range of EL4/A2/2 peptide were as sensitive to lysis by E7/A2/2-directed CTL (Fig. 3d) as correspondingly pulsed EL4.A2 cells (Fig. 2d). Additionally, we confirmed that the murine CTLs directed to E7/A2/2 peptide were capable of HLA A*0201-restricted lysis of human E7-expressing cervical carcinoma CaSki cells, in which E7 antigen-processing occurs in the context of human antigen-processing machinery. Together the data suggest that E7 is not processed by the mouse antigen-processing machinery for presentation of E7/A2/1 and E7/A2/2 HLA A*0201-restricted CTL epitopes.

Figure 3.

Figure 3

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(a–d) Splenocytes from A2.1Kb mice immunized with E7/A2/1(a), E7/A2/2 (b, d) or E7/Db (c)peptides, were restimulated in vitro with cognate peptide and reacted with unpulsed EL4.A2.E7 and EL4.A2 target cells (a–c) or EL4.A2.E7 target cells pulsed with peptide E7/A2/2 (e)as indicated, in a 4-hr 51Cr-release assay. (e) Splenocytes from A2.1Kb mice and C57BL/6 mice immunized with E7/A2/2 peptide were restimulated in vitro and reacted with CaSki cells in a 4-hr 51Cr-release assay.

Murine CTL recognizing HLA A*0201-restricted CTL epitopes are not restimulated by murine A2.1Kb cells endogenously expressing E7

We asked whether murine cells endogenously expressing whole E7 protein would process and present epitopes at the cell surface in the context of A2.1 capable of restimulating A2.1-restricted CTL precursors primed by peptide immunization. Splenocytes from E7/A2/2 peptide epitope-immunized A2.1Kb mice were divided into two aliquots. The first aliquot was restimulated in vitro with irradiated EL4.A2.E7 cells. These restimulated splenocytes completely failed to kill E7/A2/2 peptide-pulsed EL4.A2 target cells [Fig. 4(a)(i)]. The second aliquot was restimulated in vitro with E7/A2/2 peptide. These peptide-restimulated splenocytes efficiently killed E7/A2/2 peptide-pulsed EL4.A2 targets cells [Fig. 4(a)(ii)]. These data indicate that EL4.A2.E7 cells fail to restimulate A2-restricted E7/A2/2-directed CTL precursors.

Figure 4.

Figure 4

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(a) (i,ii) Splenocytes from E7/A2/2 peptide-immunized A2.1Kb mice were restimulated with irradiated EL4.A2.E7 cells (i), or with E7/A2/2 peptide (ii), and reacted with peptide-pulsed EL4.A2 target cells as indicated. (iii,iv) Splenocytes from E7/Db peptide-immunized mice were restimulated with irradiated EL4.A2.E7 cells (iii) or with E7/Db peptide (iv), and reacted with peptide-pulsed EL4.A2 target cells as indicated, in a 4-hr 51Cr-release assay. (b)Splenocytes from E7.GST-immunized A2.1Kb mice were (i) restimulated in vitro with a mix of E7/A2/1 and E7/A2/2 peptides and reacted with EL4.A2 target cells pulsed with E7/A2/1 peptide or E7/A2/2 peptide, as indicated or (ii) restimulated in vitro with E7/Db peptide and reacted with EL4.A2 target cells pulsed with E7/Db peptide, and with EL4.A2.E7 target cells, in a 4-hr 51Cr-release assay.

To confirm that the EL4.A2.E7 cells were competent for antigen processing and presentation in the restimulation process, splenocytes from E7/Db peptide-immunized mice were similarly divided into two aliquots. The first aliquot was restimulated with EL4.A2.E7 cells. These restimulated splenocytes killed E7/Db-pulsed EL4.A2 target cells through the Db-restricted pathway [Fig. 4(a)(iii)] as efficiently as effectors from the second aliquot which were restimulated with E7/Db peptide [Fig. 4(a)(iv)].

Together, these data indicate that E7 protein was processed for the presentation of the H-2Db-restricted CTL epitope, but not for presentation of the HLA A*0201-restricted CTL epitope during restimulation of primed E7-directed CTL precursors by murine cells expressing E7 and the A2.1- and H-2Db-restriction elements.

Immunization of A2.1Kb mice with E7 protein elicits CTL to Db-restricted, but not HLA A*0201-restricted, CTL epitopes

We asked whether E7 epitopes would be processed to generate HLA A*0201-restricted CTL responses from whole E7 protein given as immunogen to A2.1 Kb mice. Splenocytes from A2.1Kb mice immunized with E7/GST protein and restimulated with an equimolar mix of E7/A2/1 peptide and E7/A2/2 peptide failed to kill EL4.A2 target cells pulsed with E7/A2/1 peptide or E7/A2/2 peptide, respectively [Fig. 4(b)(i)] In contrast, splenocytes from identically immunized A2.1Kb mice restimulated with E7/Db peptide specifically killed EL4.A2 target cells pulsed with E7/Db peptide, and EL4.A2.E7 target cells [Fig. 4(b)(ii)]. In further experiments, splenocytes from mice immunized with E7/GST and restimulated with an equimolar mix of E7/Db, E7/A2/1 and E7/A2/2 peptides specifically killed EL4.A2 cells pulsed with E7/Db peptide, but not EL4.A2 target cells pulsed with E7/A2/1 peptide or E7/A2/2 peptide (not shown).

These data indicate that although exogenously administered E7 immunogen is processed to generate and present the immunogenic E7/Db-restricted CTL epitope, which primes CTL precursors for restimulation and lytic function, it is not processed to present E7 HLA*0201-restricted CTL epitopes, in A2.1Kb transgenic mice.

Immunization of A2.1Kb mice with Db-restricted E7 epitope peptide, but not with HLA A*0201-restricted E7 epitope peptide protects against E7- and A2.1-expressing murine tumour

Several laboratories have shown that immunization with peptide containing the E7/Db-restricted CTL epitope protects mice against challenge with E7-expressing H-2b tumour.13,14 Protection is mediated by CD8+ CTLs, and in CD4-depleted mice, CD8+ cells alone are sufficient to mediate the response.14 We asked whether immunization of A2.1Kb mice with E7/A2/2 peptide epitope, which elicits E7/A2/2-directed CTL, would protect mice against challenge with an E7- and A2-expressing TC1-A2 tumour given 2 weeks later (Fig. 5a). We derived the TC1-A2 tumour by transfection of the E7-expressing TC-1 H-2b epithelial tumour line25 with A2.1Kb plasmid. Mice immunized with E7/A2/2 peptide succumbed to TC1-A2 tumour almost as readily as control mice immunized with PBS. In contrast, mice immunized with E7/Db peptide were totally protected (Fig. 5a). We demonstrated that effector CTL from E7/Db-immunized mice but not effector CTL from E7/A2/2-immunized mice specifically lysed TC1-A2 targets cells (Fig. 5b). To ensure that lack of TC1-A2 tumour protection by E7/A2/2 immunization was not due to loss of A2.1 in vivo, we confirmed persistence and functional expression of HLA A*0201 α1- and α2-chains in TC1-A2 cells obtained from biopsies taken 20 days after tumour challenge by PCR and by susceptibility to lysis by E7/A2/2-directed CTL when the TC1-A2 cells were pulsed with E7/A2/2 peptide (not shown).

Figure 5.

Figure 5

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(a) A2.1Kb mice were immunized twice at an interval of 3 weeks with E7/A2/2 peptide, E7/Db peptide, or with PBS as indicated. Two weeks after the second immunization, mice were injected s.c. with 2×105 TC1-A2 tumour cells. The number of mice free of tumour nodules was quantified at intervals to 60 days after tumour inoculation. (b) Splenocytes from E7/Db-immunized mice and from E7/A2/2-immunized mice were restimulated with cognate peptide and reacted with TC1.A2 cells in a 4-hr 51Cr-release assay.

Overall, these results are consistent with the notion that while endogenous E7 in the tumour can be processed for presentation of the H-2Db-restricted E7 epitope, which renders the tumour susceptible to control by E7/Db-directed CTL, E7 in the tumour cannot be processed for the presentation of the HLA A*0201-restricted E7 epitope, which renders the tumour resistant to control by E7/A2/2-directed CTLs.

Immunization with HLA A*0201-restricted epitopes of E7 in an alternative protein configuration does not elicit A2-restricted, E7-directed CTLs

A number of laboratories have shown that A2.1Kb transgenic mice can process HLA A*0201-restricted epitopes out of various whole protein immunogens,4,6 i.e. the failure of E7 to be processed and presented for generation of E7-directed CTLs restricted through HLA A*0201 is not due to a constitutive inability of A2 transgenic mice to process and present HLA A*0201-restricted epitopes from whole protein. We therefore asked whether placing the HLA A*0201-restricted E7 epitopes in a different topographical site to their natural positions in the whole E7 molecule might render them susceptible to processing and presentation. We immunized mice with a DNA polytope (pCCV2000) vaccine encoding the E7/A2/1 and E7/A2/2 epitopes, the E7/Db epitope, and the influenza/A2 epitope (Fig. 6a). Splenocytes from pCCV2000-immunized A2.1Kb mice restimulated with cognate peptide were strongly cytotoxic for influenza/A2 peptide-pulsed EL4.A2 cells, and for E7/Db-pulsed EL4.A2 cells, indicating that these epitopes had been processed out of the protein product of pCCV2000 DNA [Fig. 6(b)(i),(iv)]. However, restimulated splenocytes failed to kill either E7/A2/1- or E7/A2/2-pulsed EL4.A2 cells [Fig. 6(b)(ii),(iii)]. Splenocytes from mice immunized with an equimolar mix of peptides expressed by the pCCV2000 polytope (influenza/A2, E7/A2/1, E7/A2/2 and E7/Db), and restimulated separately with individual peptides killed EL4.A2 cells pulsed with the cognate peptide (not shown).

Figure 6.

Figure 6

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(a) Peptide domain structure of the pCCV2000 gene product, showing the endoplasmic reticulum signal sequence, the PADRE ‘universal’ T-helper epitope, and CTL epitopes relevant to the present study. (b) (i–iv); splenocytes from pCCV2000 DNA immunized A2.1Kb mice were restimulated with E7/Db (i), E7/A2/1 (ii), E7/A2/2 (iii), or influenza/A2 (iv) peptides and reacted with EL4.A2 cells pulsed with peptide as indicated in a 4-hr 51Cr-release assay. (v–viii), splenocytes from pCCV2000-immunized A2.1Kb mice (v,vi), E7/A2/2 peptide immunized mice (vii), or influenza/A2 peptide immunized mice (viii), were restimulated with E7/A2/2 peptide (v,vii) or with influenza/A2 peptide (vi,viii), and reacted with PE-labelled E7/A2/2-tetramer44 (v,vii) or influenza/A2-tetramer44 (vi,viii) and FITC-labelled anti-CD8, and visualized by dual-colour FACS analysis.(c) (ix,x); splenocytes from pCCV2000 DNA immunized HHD mice were restimulated with syngeneic lymphoblasts loaded with E7/A2/2 (ix), or influenza/A2 (x) peptides, and reacted with EL4S3– RobHHD cells pulsed with peptide as indicated, in a 4-hr 51Cr-release assay.

We also examined these CTL effector populations for binding to MHC class 1 tetramers. Epitope-specific tetramers provide a more sensitive tool to quantify CD8+ T cells bearing cognate T-cell receptors. Approximately 8% of influenza/A2-restimulated CD8+ T cells from A2.1Kb transgenic mice immunized with pCCV200 reacted with influenza/A2 tetramer [Fig. 6(b)(vi)], correlating with the CTL efficacy of this cell population against influenza/A2-pulsed target cells [Fig. 6(b)(iv)]. A similar number of influenza/A2 tetramer-reactive CD8+ T cells were seen in specifically restimulated splenocytes from mice immunized with influenza/A2 peptide [Fig. 6(b)(viii)], indicating the efficacy of pCCV2000 DNA vaccine for delivering HLA A*0201-restricted CTL epitopes. However, virtually no E7/A2/2-tetramer-positive CD8+ T cells were seen in E7/A2/2-restimulated splenocytes from pCCV2000 immunized A2.1Kb mice. [Fig. 6(b)(v)]. This contrast with a E7/A2/2-tetramer-positive population was seen in specifically restimulated CD8+ splenocytes from E7/A2/2-peptide immunized mice [Fig. 6(b)(vii)].

Overall, these data indicate that in the alternative topographic location provided by pCCV2000 neither E7/A2/1 nor E7/A2/2 epitopes were processed and presented from the pCCV2000 protein product for priming of E7-directed A2-restricted CD8+ T-cell responses.

HHD A2.1 transgenic mice, which have been rendered incapable of endogenous murine MHC-class I-restricted CTL responses, are generally superior to A2.1Kb transgenic mice which still express H-2 class I molecules, in the generation of A2-restricted CTL responses following immunization with A2-restricted epitopes.21 In order to inquire further whether the HLA A*0201-restricted E7 CTL epitopes in the context of pCCV2000 vaccine could be processed and presented, we immunized HHD mice with pCCV2000, restimulated the splenocytes with peptide, and measured CTL responses. Specifically restimulated spleen cells from HHD mice immunized with pCCV2000 failed to kill EL4/A2/2-pulsed EL4S3– RobHHD target cells. In contrast, influenza/A2-pulsed EL4S3– RobHHD targets were killed with high efficiency [Fig. 6(c)(ix, x)]. Together, these data are consistent with the notion that A2 transgenic mice (even those maximized for HLA A*0201-restricted responses) are unable to process and present the E7 HLA A*0201-restricted epitopes encoded by the DNA polyepitope.

Discussion

HLA A*0201 transgenic mice have been widely used to assess the in vivo immunogenicity of potential CTL epitopes identified as peptides capable of binding to HLA A*0201 molecules.6,30,31 The overall agreement between immunogenicity of the peptides tested either in vivo in HLA transgenic mice or in vitro in primary CTL induction experiments with human PBMCs, suggests that both approaches are applicable to the identification of potential CTL epitopes from antigens of choice. Indeed, the data suggesting an extensive overlap between TCR repertoires of mouse and human support the use of HLA trangenic mice for the identification of potential human CTL epitopes.31,32 In many transgenic mouse studies, the CTL obtained were tested for specificity on MHC-matched target cells coated with peptide. However, recognition of endogenously processed antigen is necessary, since only that provides evidence for induction of immunologically relevant T cells.1

The HLA A*0201-restricted E7 CTL epitopes used in this study were identified as having high binding affinity for the HLA A*0201 molecules7 from a set of all possible nonomer peptides spanning the HPV16 E7 molecule. The epitopes were shown to be immunogenic when used as peptides to immunize HLA A*0201 transgenic mice and to induce primary CTL responses in peripheral blood mononuclear cells in vitro from HLA A*0201+ donors.19 Human CTL clones specific for these peptides lysed the HPV16 E7-expressing, HLA A*0201-expressing cervical carcinoma cell line CaSki.19 These data suggest that in humans these peptides are likely to represent naturally processed epitopes of HPV16 E7.

In the present study, we examined the ability of two HLA A*0201-restricted E7 CTL epitopes to be processed and presented from whole E7 in the context of the mouse antigen-processing machinery. We show that immunization of A2.1Kb transgenic mice with whole E7 protein will not generate E7/A2/1-directed or E7/A7/2-directed CTL responses, although responses to the murine E7/Db-restricted epitope were generated (Fig. 4). This result shows that processing of E7 by murine antigen-presenting cells is not capable of presenting these HLA A*0201-restricted CTL epitopes for recognition by appropriate precursors in the murine T-cell repertoire, while confirming that E7 is processed for the generation of the H-2b-restricted CTL epitope. Additionally, we show that CTL induced by immunization of HLA A*0201 transgenic mice with peptides representing the HLA A*0201-restricted CTL epitopes were incapable of killing EL4.A2.E7 target cells which endogenously express E7 and which demonstrably present E7 for killing by CTL from mice immunized with peptide representing the E7 H-2b-restricted CTL epitope (Fig. 3). Furthermore, HLA A*0201-expressing EL4 cells are capable of processing HLA A*0201-restricted CTL epitopes from endogenously expressed proteins other than E7,4 indicating that the inability to present E7 HLA A*0201 CTL epitopes is not due to a constitutive inability of these cells to process and present HLA A*0201 epitopes per se. In agreement with these findings we show that EL4/A2/E7 cells are incapable of restimulating CTL precursors directed to either of the HLA A*0201-restricted E7 CTL epitopes, although CTL precursors directed to the H-2b-restricted E7 epitope were restimulated (Fig. 4).

It is known that the topographic site of an epitope within a protein may influence the epitope's immunogenicity.2 However, arranging the E7/A2/1and E7/A2/2 E7 epitopes to an alternative location within a protein using a DNA vaccine approach, did not result in the in vivo activation of epitope-specific T cells, either measured functionally in CTL assays, or by more sensitive quantification of epitope-specific MHC class 1 tetramer binding (Fig. 6a, b). Since tetramer binding depends on interaction between TCR and the α1 α2 domains of class 1 with cognate peptide bound in the groove, tetramers composed of native HLA A2.1 may be used to quantify T cells from A2.1Kb transgenic mice which have been educated on a chimeric class 1 containing the murine (Kb) α3 domain.33

The possibility that an immunodominance effect exerted by influenza/A2 and E7/Db epitopes over the E7/A2/1 and E7/A2/2 epitopes is unlikely since immunization with the E7 A2-restricted epitopes and the influenza/A2 and E7/Db epitopes as a peptide mix elicited good CTL to each. Furthermore, the polyepitope DNA vaccine approach tends to minimize the immunodominance effects otherwise seen between constituent epitopes.34 In support of the concept that HLA A*0201-restricted CTL epitopes are not processed out of whole E7 in mice, we found that immunization with HLA A*0201 epitope peptide (which generates CTL) failed to control the growth of an E7- and HLA A2-expressing tumour, although tumour growth was effectively controlled through the H-2b pathway by immunization with the Db-restricted E7 epitope peptide (Fig. 5).

The question which arises is why HLA A*0201 transgenic mice are incapable of processing whole E735 for the generation of the HLA A*0201 E7 CTL epitopes used in the present study. The literature remains unclear whether all components of the antigen presentation machinery function normally across species, or for all peptide–HLA combinations. There are multiple levels in the antigen processing and presentation pathway at which perturbation could result in failure to present the E7 epitopes, from digestion of whole protein by endopeptidases, to translocation and insertion of the peptide-loaded class 1 molecule into the cell surface. Species-specific generation of peptide epitopes may preclude E7/A2/1 and E7/A2/2 peptides in mice; indeed this phenomenon has been implicated as an explanation of differential presentation of a HLA-A3 antigenic epitope from influenza A nucleoprotein by mouse and human cells.36 However, if the HLA A*0201 E7 peptides were generated, selective impaired peptide translocation into the endoplasmic reticulum lumen by murine transporter associated with antigen processing (TAP) could possibly preclude epitope presentation. Alternatively, any of the multiple molecular interactions which collaborate to load peptides efficiently into the antigen-binding cleft of the class 1 molecules, including association of empty class 1/β2-microglobulin complexes with calnexin, calreticulin and ER-60 chaperones,37–39 may be sub-optimal in the case of E7 HLA A*0201 peptides. Ultimately, efficient peptide loading of MHC class 1 molecules is critically dependent on the endoplasmic reticulum glycoprotein tapasin, which itself may operate at several levels to ensure adequate stabilization and expression of peptide-loaded class 1 complexes, including bridging of class 1 molecules with TAP5 and direct effects on the peptide loading process.5,40

The possibility that competition between murine H-2b and human A2 molecules in the endoplasmic reticulum for access to TAP/tapasin complexes is an explanation for the lack of presentation of processed HLA A*0201 E7 epitopes is unlikely, because these epitopes were not processed and presented in HHD mice (Fig. 6) which lack processing through the H-2 pathway.

In summary, HLA-class 1 transgenic mice are widely used for studies of human CTL epitope presentation in murine models of human disease. These models assume conserved antigen presentation function including physiological peptide generation and selection by human class 1 molecules in murine antigen-presenting cells. While this clearly occurs in some instances2–4,41, our data indicate that there are limitations with regard to the E7 CTL epitopes which although processed from E7 in HLA A*0201+ humans19,20 are not processed in HLA A*0201 transgenic mice.

Acknowledgments

Dr Linda Sherman and the Scripps Institute gave permission to use the A2.1Kb mice. Dr F. Lemonnier gave permission to use the HHD mice. Greg Bryson performed the FACS analyses. Dr Germain Fernando provided the E7.GST fusion protein. Dr Scott Thomson made the pCCV2000 DNA polytope. Dr TC Wu provided TC-1 cells. We thank Ms Donna West and her staff for excellent animal husbandry. The work was supported by grants from the National Health and Medical Research Council (NHMRC, Australia) and Queensland Cancer Fund.

References

  • 1.Alexander J, Oseroff C, Sidney J, et al. Derivation of HLA-A11/Kb transgenic mice: functional CTL repertoire and recognition of human A11-restricted CTL epitopes. J Immunol. 1997;159:4753–61. [PubMed] [Google Scholar]
  • 2.Ishioka GY, Fikes J, Hermanson G, et al. Utilization of MHC class I transgenic mice for development of minigene DNA vaccines encoding multiple HLA-restricted CTL epitopes. J Immunol. 1999;162:3915–25. [PubMed] [Google Scholar]
  • 3.Loirat D, Lemonnier FA, Michel ML. Multiepitopic HLA A*0201 restricted immune response against Hepatitis B surface antigen after DNA based immunisation. J Immunol. 2000;165/8:4748–55. doi: 10.4049/jimmunol.165.8.4748. [DOI] [PubMed] [Google Scholar]
  • 4.Vitiello A, Marchesini D, Furze J, Sherman LA, Chesnut RW. Analysis of the HLA-restricted influenza-specific cytotoxic T lymphocyte response in transgenic mice carrying a chimeric human-mouse class I major histocompatibility complex. J Exp Med. 1991;173:1007–15. doi: 10.1084/jem.173.4.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Peh CA, Laham N, Burrows SR, Zhu Y, McCluskey J. Distinct functions of tapasin revealed by polymorphism in MHC class I peptide loading. J Immunol. 2000;164:292–9. doi: 10.4049/jimmunol.164.1.292. [DOI] [PubMed] [Google Scholar]
  • 6.Geluk A, van Meijgaarden KE, Franken KL, Drijfhout JW, D'Souza S, Necker A, Huygen K. Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognised by HLA A*0201 restricted CD8 T cells in HLA transgenic mice and humans. J Immunol. 2000;165:6463–71. doi: 10.4049/jimmunol.165.11.6463. [DOI] [PubMed] [Google Scholar]
  • 7.Kast WM, Brandt RM, Sidney J, Drijfhout JW, Kubo RT, Grey HM, Melief CJ, Sette A. Role of HLA-A motifs in identification of potential CTL epitopes in human papillomavirus type 16, E6 and E7 proteins. J Immunol. 1994;152:3904–12. [PubMed] [Google Scholar]
  • 8.Theobald M, Biggs J, Hernandez J, Lustgarten J, Labadie C, Sherman LA. Tolerance to p53 by A2.1-restricted cytotoxic T lymphocytes. J Exp Med. 1997;185:833–41. doi: 10.1084/jem.185.5.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Yang S, Linette GP, Longerich S, Roberts BL, Haluska FG. HLA-A2.1K (b) transgenic murine dendritic cells transduced with an adenovirus gp100 process the same A2.1-restricted peptide epitopes as human antigen presenting cells and elicit A2.1 restricted peptide specific CTL. Cellular Immunol. 2000;204:29–37. doi: 10.1006/cimm.2000.1695. [DOI] [PubMed] [Google Scholar]
  • 10.von Knebel Doeberitz M, Rittmuller C, Aengeneyndt F, Jansen Durr P, Spitkovsky D. Reversible repression of papillomavirus oncogene expression in cervical carcinoma cells: consequences for the phenotype and E6-p53 and E7–pRB interactions. J Virol. 1994;68:2811–21. doi: 10.1128/jvi.68.5.2811-2821.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Tindle RW. Human papillomavirus vaccines for cervical cancer. Curr Opin Immunol. 1996;8:643–50. doi: 10.1016/s0952-7915(96)80080-x. [DOI] [PubMed] [Google Scholar]
  • 12.Wang TL, Ling M, Shih IM, et al. Intramuscular administration of E7-transfected dendritic cells generates the most potent E7-specific anti-tumor immunity. Gene Ther. 2000;7:726–33. doi: 10.1038/sj.gt.3301160. [DOI] [PubMed] [Google Scholar]
  • 13.Feltkamp MC, Vreugdenhil GR, Vierboom MP, Ras E, van der Burg SH, ter Schegget J, Melief CJ, Kast WM. Cytotoxic T lymphocytes raised against a subdominant epitope offered as a synthetic peptide eradicate human papillomavirus type 16-induced tumors. Eur J Immunol. 1995;25:2638–42. doi: 10.1002/eji.1830250935. [DOI] [PubMed] [Google Scholar]
  • 14.Fernando GJ, Stewart TJ, Tindle RW, Frazer IH. Th2-type CD4+ cells neither enhance nor suppress antitumor CTL activity in a mouse tumor model. J Immunol. 1998;161:2421–7. [PubMed] [Google Scholar]
  • 15.Ji H, Chang EY, Lin KY, Kurman RJ, Pardoll DM, Wu TC. Antigen-specific immunotherapy for murine lung metastatic tumors expressing human papillomavirus type 16, E7 oncoprotein. Int J Cancer. 1998;78:41–5. doi: 10.1002/(sici)1097-0215(19980925)78:1<41::aid-ijc8>3.0.co;2-x. 10.1002/(sici)1097-0215(19980925)78:1<41::aid-ijc8>3.3.co;2-k. [DOI] [PubMed] [Google Scholar]
  • 16.Doan T, Chambers M, Street M, Fernando GJ, Herd K, Lambert P, Tindle R. Mice expressing the E7 oncogene of HPV16 in epithelium show central tolerance, and evidence of peripheral anergising tolerance, to E7-encoded cytotoxic T-lymphocyte epitopes. Virology. 1998;244:352–64. doi: 10.1006/viro.1998.9128. [DOI] [PubMed] [Google Scholar]
  • 17.Rosenberg SA, Yannelli JR, Yang JC, et al. Treatment of patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and interleukin 2 [see comments] J Natl Cancer Inst. 1994;86:1159–66. doi: 10.1093/jnci/86.15.1159. [DOI] [PubMed] [Google Scholar]
  • 18.Rosenberg SA, Zhai Y, Yang JC, et al. Immunizing patients with metastatic melanoma using recombinant adenoviruses encoding MART-1 or gp100 melanoma antigens. J Natl Cancer Inst. 1998;90:1894–900. doi: 10.1093/jnci/90.24.1894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Ressing ME, Sette A, Brandt RM, et al. Human CTL epitopes encoded by human papillomavirus type 16, E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLA-A*0201-binding peptides. J Immunol. 1995;154:5934–43. [PubMed] [Google Scholar]
  • 20.Ressing ME, Offringa R, Toes RE, Ossendorp F, de Jong JH, Brandt RM, Kast WM, Melief CJ. Immunotherapy of cancer by peptide-based vaccines for the induction of tumor-specific T cell immunity. Immunotechnology. 1996;2:241–51. doi: 10.1016/s1380-2933(96)00057-7. 10.1016/s1380-2933(96)00057-7. [DOI] [PubMed] [Google Scholar]
  • 21.Firat H, Garcia-Pons F, Tourdot S, et al. H-2 class 1 knockout, HLA A2.1 transgenic mice: a versatile animal model for preclinical evaluation of anti tumour therapeutic strategies. Eur J Immunol. 1999;29/10:3112–21. doi: 10.1002/(SICI)1521-4141(199910)29:10<3112::AID-IMMU3112>3.0.CO;2-Q. [DOI] [PubMed] [Google Scholar]
  • 22.Fernando GJ, Murray B, Zhou J, Frazer IH. Expression, purification and immunological characterization of the transforming protein E7, from cervical cancer-associated human papillomavirus type 16. Clin Exp Immunol. 1999;115:397–403. doi: 10.1046/j.1365-2249.1999.00813.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Tindle RW, Croft S, Herd K, Malcolm K, Geczy AF, Stewart T, Fernando GJ. A vaccine conjugate of ‘ISCAR’ immunocarrier and peptide epitopes of the E7 cervical cancer-associated protein of human papillomavirus type 16 elicits specific Th1- and Th2-type responses in immunized mice in the absence of oil-based adjuvants. Clin Exp Immunol. 1995;101:265–71. doi: 10.1111/j.1365-2249.1995.tb08349.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Pascolo S, Bervas N, Ure JM, Smith AG, Lemonnier FA, Perarnau B. HLA-A2-restricted education and cytolytic activity of CD8+ lymphocytes from β2 microglobulin (β2m) HLA-A2.1 monochain transgenic H-2Dbβ2m double knockout mice. J Exp Med. 1997;185:2043–51. doi: 10.1084/jem.185.12.2043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Lin KY, Guarnieri FG, Staveley O-KF, Levitsky HI, August JT, Pardoll DM, Wu TC. Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res. 1996;56:21–6. [PubMed] [Google Scholar]
  • 26.Fossum C, Bergstrom M, Lovgren K, Watson DL, Morein B. Effect of iscoms and their adjuvant moiety (matrix) on the initial proliferation and IL-2 responses: comparison of spleen cells from mice inoculated with iscoms and/or matrix. Cell Immunol. 1990;129:414–25. doi: 10.1016/0008-8749(90)90217-f. [DOI] [PubMed] [Google Scholar]
  • 27.Herd K, Fernando GJ, Dunn LA, Frazer IH, Lambert P, Tindle RW. E7 oncoprotein of human papillomavirus type 16 expressed constitutively in the epidermis has no effect on E7-specific B- or Th-repertoires or on the immune response induced or sustained after immunization with E7 protein. Virology. 1997;231:155–65. doi: 10.1006/viro.1997.8491. [DOI] [PubMed] [Google Scholar]
  • 28.Thomson SA, Sherritt MA, Medveczky J, Elliott SL, Moss DJ, Fernando GJ, Brown LE, Suhrbier A. Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination. J Immunol. 1998;160:1717–23. [PubMed] [Google Scholar]
  • 29.Alexander J, Fikes J, Hoffman S, et al. The optimization of helper T lymphocyte (HTL) function in vaccine development. Immunol Res. 1998;18:79–92. doi: 10.1007/BF02788751. [DOI] [PubMed] [Google Scholar]
  • 30.Schell TD, Lippolis JD, Tevethia SS. Cytotoxic T lymphocytes from HLA A2.1 transgenic mice define a potential human epitope from simian virus 40 large T antigen. Cancer Res. 2001;61/3:873–9. [PubMed] [Google Scholar]
  • 31.Wentworth PA, Vitiello A, Sidney J, Keogh E, Chesnut RW, Grey H, Sette A. Differences and similarities in the A2.1-restricted cytotoxic T cell repertoire in humans and human leukocyte antigen-transgenic mice. Eur J Immunol. 1996;26:97–101. doi: 10.1002/eji.1830260115. [DOI] [PubMed] [Google Scholar]
  • 32.Ressing ME, van Driel WJ, Brandt RM, et al. Detection of T helper responses, but not of human papillomavirus specific cytotoxic T lymphocyte responses after peptide vaccination with cervical carcinoma. J Immunotherapy. 2000;23/2:255–66. doi: 10.1097/00002371-200003000-00010. [DOI] [PubMed] [Google Scholar]
  • 33.Sherman LA, Hesse SV, Irwin MJ, la Face D, Peterson P. Selecting T cell receptors with high affinity for self-MHC by decreasing the contribution of CD8. Science. 1992;258:815–18. doi: 10.1126/science.1439792. [DOI] [PubMed] [Google Scholar]
  • 34.Thomson SA, Sherritt MA, Medveczky J, Elliott SL, Moss DJ, Fernando GJ, Brown LE, Suhrbier A. Delivery of multupple CD8 cytotoxic T cell epitopes by DNA vaccination. J Immunol. 1998;160/4:1717–23. [PubMed] [Google Scholar]
  • 35.Bauer M, Wagner H, Lipford GB. HPV type 16 protein E7 HLA-A2 binding peptides are immunogenic but not processed and presented. Immunol Lett. 2000;71:55–9. doi: 10.1016/s0165-2478(99)00170-4. [DOI] [PubMed] [Google Scholar]
  • 36.Braud VM, McMichael AJ, Cerundolo V. Differential processing of influenza nucleoprotein in human and mouse cells. Eur J Immunol. 1998;28:625–35. doi: 10.1002/(SICI)1521-4141(199802)28:02<625::AID-IMMU625>3.0.CO;2-I. [DOI] [PubMed] [Google Scholar]
  • 37.Degen E, Cohen-Doyle MJ, Williams DB. Efficient dissociation of the p88 chaperone from major histocompatibility complex class 1 molecules requires both beta-2 microglobulin and peptide. J Exp Med. 1992;175:1653–61. doi: 10.1084/jem.175.6.1653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Lindquist JA, Jensen ON, Mann M, Hammerling GJ. ER-60, a chaperone with thiol-dependent reductase activity involved in MHC class I assembly. EMBO J. 1998;17:2186–95. doi: 10.1093/emboj/17.8.2186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Sadasivan B, Lehner PJ, Ortmann B, Spies T, Cresswell P. Roles for calreticulin and a novel glycoprotein, tapasin, in the interaction of MHC class I molecules with TAP. Immunity. 1996;5:103–14. doi: 10.1016/s1074-7613(00)80487-2. [DOI] [PubMed] [Google Scholar]
  • 40.Lehner PJ, Surman MJ, Cresswell P. Soluble tapasin restores MHC class I expression and function in the tapasin-negative cell line.220. Immunity. 1998;8:221–31. doi: 10.1016/s1074-7613(00)80474-4. [DOI] [PubMed] [Google Scholar]
  • 41.Kuon W, Lauster R, Bottcher U, et al. Recognition of chlamydial antigen by HLA-B27-restricted cytotoxic T cells in HLA-B*2705 transgenic CBA (H-2k) mice. Arthritis Rheum. 1997;40:945–54. doi: 10.1002/art.1780400524. [DOI] [PubMed] [Google Scholar]
  • 42.Feltkamp MC, Smits HL, Vierboom MP, et al. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16- transformed cells. Eur J Immunol. 1993;23:2242–9. doi: 10.1002/eji.1830230929. [DOI] [PubMed] [Google Scholar]
  • 43.Gotch F, Rothbard J, Howland K, Townsend A, McMichael A. Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2. Nature. 1987;326:881–2. doi: 10.1038/326881a0. [DOI] [PubMed] [Google Scholar]
  • 44.Tindle RW, Herd K, Doan T, Bryson G, Leggatt GR, Lambert P, Frazer IH, Street M. Non-specific down-regulation of CD8+ T cell responses in mice expressing Human Papillomavirus Type from the keratin-14 promoter. J Virol. 2001;75:5985–97. doi: 10.1128/JVI.75.13.5985-5997.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]

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